Current protocols essential laboratory techniques / ed. by S.R.Gallagher, E.A.Wiley. - Hoboken: Wiley-Blackwell, 2012. - various pagings: ill. - Incl. bibl. ref. and index. - ISBN 978-0-470-94241-3  Место хранения:   033 | Бурятский научный центр | Улан-Удэ | Центральная научная библиотека

Оглавление / Contents

Foreword ....................................................... xi Preface ...................................................... xiii About the Second Edition and Online Access ................ xiii How to Use This Manual ..................................... xiv Acknowledgments ............................................ xvi Access to Current Protocols Essential Laboratory Techniques Online .................................................... xvii Contributors .................................................. xxi Common Conversion Factors ................................... xxiii Introduction ............................................. xxiii Converting Units of Volume ............................... xxiii Converting Temperatures ................................... xxiv A Note About Writing Units of Measure .................... xxvii Internet Resources ...................................... xxviii Combining Techniques to Answer Molecular Questions ........... xxix Introduction .............................................. xxix Nucleic Acids ............................................. xxix Proteins .................................................. xxxi Whole Cells and Subcellular Structures ................... xxxii General .................................................. xxxii Internet Resources ...................................... xxxiii 1 Volume/Weight Measurement 1.1 Volume Measurement Overview and Principles ............................. 1.1.1 Micropipettors ...................................... 1.1.5 Pipets .............................................. 1.1.8 Volumetric Containers .............................. 1.1.11 Burets and Graduated Cylinders ..................... 1.1.12 Cleaning Volumetric Apparatus ...................... 1.1.12 Literature Cited ................................... 1.1.14 Internet Resources ................................. 1.1.14 1.2 Weight Measurement Overview and Principles ............................. 1.2.1 Strategic Planning .................................. 1.2.3 Safety Considerations ............................... 1.2.9 Protocols .......................................... 1.2.10 Basic Protocol 1: Measuring Mass Using a Top- Loading Balance ................................. 1.2.10 Basic Protocol 2: Measuring Mass Using an Analytical Balance .............................. 1.2.10 Understanding Results .............................. 1.2.10 Troubleshooting .................................... 1.2.11 Literature Cited ................................... 1.2.11 Internet Resources ................................. 1.2.11 2 Concentration Measurement 2.1 Spectrophotometry Overview and Principles ............................. 2.1.1 Components of a Spectrophotometer ................... 2.1.4 How a Spectrophotometer Works ....................... 2.1.8 Strategic Questions ................................ 2.1.13 Strategic Planning ................................. 2.1.15 Safety Considerations .............................. 2.1.19 Protocols .......................................... 2.1.19 Basic Protocol 1: Preparation of a Standard Curve for Dipicolinic Acid (DPA) ................ 2.1.19 Basic Protocol 2: Determination of a Standard Curve for Dipicolinic Acid (DPA) and Measurement of DPA Concentration in Unknown Samples ......................................... 2.1.21 Support Protocol: Optimizing Spectrophotometer Settings ........................................ 2.1.26 Understanding Results .............................. 2.1.27 Troubleshooting .................................... 2.1.27 Literature Cited ................................... 2.1.28 Internet Resources ................................. 2.1.28 2.2 Quantitation of Nucleic Acids and Proteins Overview and Principles ............................. 2.2.1 Strategic Questions ................................. 2.2.7 Strategic Planning .................................. 2.2.8 Protocols: Nucleic Acid Quantification ............. 2.2.13 Basic Protocol 1: Traditional Detection of Nucleic Acids Using Absorption Spectroscopy ..... 2.2.13 Basic Protocol 2: Microvolume Detection of Nucleic Acids Using Absorption Spectroscopy ..... 2.2.16 Alternate Protocol 1: DNA Detection Using the DNA-Binding Fluorochrome Hoechst 33258 .......... 2.2.19 Alternate Protocol 2: DNA and RNA Detection with Ethidium Bromide Fluorescence .............. 2.2.20 Alternate Protocol 3: DNA Detection Using PicoGreen dsDNA Quantitation Reagent ............ 2.2.21 Protocols: Protein Quantification .................. 2.2.25 Basic Protocol 3: Lowry Protein Assay ........... 2.2.25 Alternate Protocol 4: Lowry Protein Assay, Reduced Volume .................................. 2.2.26 Support Protocol: Deoxycholate-Trichloroacetic Acid (DOC-TCA) Sample Precipitation for Removal of Interfering Compounds and Sample Concentration ................................... 2.2.27 Basic Protocol 4: BCA Protein Assay ............. 2.2.27 Basic Protocol 5: Coomassie Blue Protein Assay (Bradford Assay) ................................ 2.2.28 Basic Protocol 6: Traditional UV Spectrophotometry ............................... 2.2.29 Alternate Protocol 5: Protein Quantitation with UV Spectroscopy and Correction for Like- Acid Contamination ............................. 2.2.29 Basic Protocol 7: Microvolume UV Spectrophotometry ............................... 2.2.30 Protocol Common to Nucleic Acids and Proteins ...... 2.2.32 Basic Protocol 8: Gel-Based Quantitation of Proteins and Nucleic Acids ...................... 2.2.32 Reagents and Solutions ............................. 2.2.34 Understanding Results .............................. 2.2.36 Troubleshooting .................................... 2.2.37 Variations ......................................... 2.2.38 Literature Cited ................................... 2.2.38 Key References ..................................... 2.2.39 Internet Resources ................................. 2.2.39 3 Reagent Preparation 3.1 Reagent Preparation: Theoretical and Practical Discussions Reagent Preparation ................................. 3.1.1 Accuracy of Weighing and Pipetting .................. 3.1.6 Use of Calibrated pH Meters ......................... 3.1.6 Avoiding Chemical and Microbial Contamination of Reagents ............................................ 3.1.6 Preparing Reagent or Buffer Solutions ............... 3.1.7 Making Buffer Solutions ............................ 3.1.11 Literature Cited ................................... 3.1.14 Internet Resources ................................. 3.1.14 3.2 Measurement of pH Overview and Principles ............................. 3.2.1 Equipment for Measuring pH .......................... 3.2.2 pH Electrode Care ................................... 3.2.9 Measurement of pH .................................. 3.2.11 Troubleshooting .................................... 3.2.14 Internet Resources ................................. 3.2.15 3.3 Recipes for Commonly Encountered Reagents Introduction ........................................ 3.3.1 Recipes ............................................. 3.3.1 Literature Cited ................................... 3.3.12 Internet Resources ................................. 3.3.12 4 Cell Culture Techniques 4.1 Aseptic Technique Overview and Principles ............................. 4.1.1 Strategic Planning .................................. 4.1.2 Safety Considerations ............................... 4.1.6 Techniques for Maintaining Aseptic Conditions ....... 4.1.7 Literature Cited ................................... 4.1.12 Internet Resources ................................. 4.1.12 4.2 Culture of Escherichia coli and Related Bacteria Overview and Principles ............................. 4.2.1 Safety Considerations ............................... 4.2.2 Commonly Used Tools ................................. 4.2.2 Reagents ............................................ 4.2.3 Culturing Bacteria on Solid Media ................... 4.2.3 Growing Bacteria in Liquid Culture .................. 4.2.6 Analysis and Purification of Plasmid DNAs .......... 4.2.11 Storage of Cultures ................................ 4.2.22 Commonly Used Bacterial Media ...................... 4.2.23 Literature Cited ................................... 4.2.27 Internet Resources ................................. 4.2.27 5 Sample Preparation 5.1 Centrifugation Introduction ........................................ 5.1.1 Definitions ......................................... 5.1.1 Rotors .............................................. 5.1.5 Literature Cited .................................... 5.1.8 5.2 Purification and Concentration of Nucleic Acids Overview and Principles ............................. 5.2.1 Strategic Questions ................................. 5.2.6 Strategic Planning .................................. 5.2.7 Safety Considerations ............................... 5.2.8 Protocols ........................................... 5.2.8 Basic Protocol 1: Phenol Extraction and Ethanol Precipitation of DNA ............................. 5.2.8 Alternate Protocol 1: Purification of Plasmid DNA Using Silica Membrane Spin Columns .......... 5.2.10 Support Protocol 1: Precipitation of DNA Using Isopropanol ..................................... 5.2.12 Support Protocol 2: Concentration of DNA Using Butanol ......................................... 5.2.13 Basic Protocol 2: Single-Step RNA Isolation from Cultured Cells or Tissues .................. 5.2.13 Alternate Protocol 2: Purification and Concentration of RNA and Dilute Solutions of DNA ............................................. 5.2.15 Reagents and Solutions ............................. 5.2.16 Understanding Results .............................. 5.2.17 Troubleshooting .................................... 5.2.19 Acknowledgments .................................... 5.2.21 Literature Cited ................................... 5.2.21 Internet Resources ................................. 5.2.22 6 Chromatography 6.1 Overview of Chromatography Overview and Principles ............................. 6.1.1 Common Types of Chromatography for Purification of Proteins ............................................ 6.1.3 Literature Cited ................................... 6.1.10 Internet Resources ................................. 6.1.10 7 Electrophoresis 7.1 Overview of Electrophoresis Overview and Principles ............................. 7.1.1 Strategic Questions ................................. 7.1.1 General Concepts .................................... 7.1.3 Visualization of Resolved Molecules ................. 7.1.5 Imaging Resolved Molecules .......................... 7.1.6 Literature Cited .................................... 7.1.7 Internet Resources .................................. 7.1.7 7.2 Agarose Gel Electrophoresis Overview and Principles .......................................... 7.2.1 Strategic Questions ................................. 7.2.3 Strategic Planning .................................. 7.2.3 Safety Considerations ............................... 7.2.5 Protocols ........................................... 7.2.6 Basic Protocol 1: DNA Agarose Gel Electrophoresis .................................. 7.2.6 Basic Protocol 2: Denaturing RNA Agarose Gel Electrophoresis ................................. 7.2.12 Reagents and Solutions ............................. 7.2.15 Understanding Results .............................. 7.2.16 Troubleshooting .................................... 7.2.19 Variations ......................................... 7.2.19 Acknowledgments .................................... 7.2.22 Literature Cited ................................... 7.2.22 Internet Resources ................................. 7.2.22 7.3 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Overview and Principles ............................. 7.3.1 Strategic Questions ................................. 7.3.7 Strategic Planning .................................. 7.3.8 Safety Considerations .............................. 7.3.10 Protocols .......................................... 7.3.11 Basic Protocol: Denaturing (SDS) Discontinuous Gel Electrophoresis: The Laemmli Gel Method ..... 7.3.11 Support Protocol 1: Casting a Gel for Use in Denaturing Discontinuous Electrophoresis ........ 7.3.15 Support Protocol 2: Calculating Molecular Mass .. 7.3.18 Support Protocol 3: Recrystallizing SDS ......... 7.3.20 Reagents and Solutions ............................. 7.3.21 Understanding Results .............................. 7.3.22 Troubleshooting .................................... 7.3.22 Variations ......................................... 7.3.26 Literature Cited ................................... 7.3.28 Key Reference ...................................... 7.3.28 7.4 Staining Proteins in Gels Overview and Principles ............................. 7.4.1 Strategic Questions ................................. 7.4.2 Strategic Planning .................................. 7.4.3 Protocols ........................................... 7.4.4 Basic Protocol 1: Coomassie Blue Staining ........ 7.4.4 Alternate Protocol 1: Rapid Coomassie Blue Staining ......................................... 7.4.5 Basic Protocol 2: Nonammoniacal Silver Staining .. 7.4.6 Alternate Protocol 2: Rapid Silver Staining ...... 7.4.7 Basic Protocol 3: Fluorescent Staining Using SYPRO Orange or Red .............................. 7.4.8 Support Protocol: Photography of SYPRO Orange or Red Fluorescently Stained Gels ............... 7.4.10 Reagents and Solutions ............................. 7.4.11 Understanding Results .............................. 7.4.12 Troubleshooting .................................... 7.4.13 Variations ......................................... 7.4.13 Literature Cited ................................... 7.4.14 Internet Resources ................................. 7.4.14 8 Blotting 8.1 Overview of Blotting General Overview .................................... 8.1.1 General Considerations .............................. 8.1.3 Southern and Northern Blotting ...................... 8.1.4 Immunoblotting ..................................... 8.1.15 Literature Cited ................................... 8.1.19 Internet Resources ................................. 8.1.20 8.2 Nucleic Acid Blotting: Southern and Northern Overview and Principles ............................. 8.2.1 Strategic Questions ................................. 8.2.4 Strategic Planning .................................. 8.2.5 Safety Considerations ............................... 8.2.7 Protocols ........................................... 8.2.7 Basic Protocol 1: Southern Blotting .............. 8.2.7 Basic Protocol 2: Northern Blotting ............. 8.2.13 Support Protocol: Assembling a Blotting Transfer Apparatus .............................. 8.2.17 eagents and Solutions .............................. 8.2.18 nderstanding Results ............................... 8.2.20 (Troubleshooting ................................... 8.2.20 ^Variations ........................................ 8.2.24 Literature Cited ................................... 8.2.24 Key References ..................................... 8.2.24 Internet Resources ................................. 8.2.24 8.3 Protein Blotting: Immunoblotting Overview and Principles ............................. 8.3.1 Strategic Questions ................................. 8.3.7 Strategic Planning .................................. 8.3.8 Protocols .......................................... 8.3.10 Basic Protocol 1: Protein Blotting with Semidry Systems ............................................ 8.3.11 Basic Protocol 2: Tank Transfer ................. 8.3.15 Alternate Protocol 1: Slot and Dot Blotting ..... 8.3.18 Support Protocol 1: Ponceau S Staining of Transferred Proteins ............................ 8.3.19 Support Protocol 2: India Ink Staining of Transferred Proteins ............................ 8.3.19 Support Protocol 3: Gold Staining of Transferred Proteins ............................ 8.3.19 Support Protocol 4: Alkali Enhancement of Protein Staining ................................ 8.3.20 Support Protocol 5: Fluorescent Protein Blot Staining of Transferred Proteins ................ 8.3.20 Support Protocol 6: Viewing and Photographing SYPRO Ruby-Stained Protein Blots ................ 8.3.22 Basic Protocol 3: Immunoprobing with Directly Conjugated Secondary Antibody ................... 8.3.23 Alternate Protocol 2: Immunoprobing with Avidin-Biotin Coupling to Secondary Antibody .... 8.3.25 Basic Protocol 4: Visualization with Chromogenic Substrates .......................... 8.3.26 Alternate Protocol 3: Visualization with Luminescent Substrates .......................... 8.3.27 Alternate Protocol 4: Fluorescent Blot Preparation and Analysis ........................ 8.3.29 Reagents and Solutions ............................. 8.3.35 Understanding Results .............................. 8.3.37 Troubleshooting .................................... 8.3.37 Variations ......................................... 8.3.39 Literature Cited ................................... 8.3.39 Internet Resources ................................. 8.3.40 8.4 Labeling DNA and Preparing Probes Overview and Principles ............................. 8.4.1 Strategic Planning ................................. 8.4.12 Safety Considerations .............................. 8.4.13 Protocols .......................................... 8.4.13 Basic Protocol 1: 5' End-Labeling of DNA with T4 Polynucleotide Kinase ........................ 8.4.13 Basic Protocol 2: Labeling DNA by Nick Translation ..................................... 8.4.15 Basic Protocol 3: Labeling DNA by Random Primed Synthesis ................................ 8.4.17 Support Protocol: Purification of Labeled Probes Using Gel-Filtration Spin Columns ........ 8.4.19 Reagents and Solutions ............................. 8.4.20 Understanding Results .............................. 8.4.20 Troubleshooting .................................... 8.4.20 Variations ......................................... 8.4.20 Literature Cited ................................... 8.4.22 Internet Resources ................................. 8.4.23 9 Microscopy 9.1 Conventional Light Microscopy Parts of the Light Microscope ....................... 9.1.1 Care and Maintenance ................................ 9.1.1 Basic Principles and Definitions .................... 9.1.4 Magnification Versus Resolution ..................... 9.1.7 Getting Comfortable ................................. 9.1.9 Finding the Object to be Viewed .................... 9.1.10 Marking the Location of an Object: Secrets of the Microscope Stage ................................... 9.1.12 A Quick Guide to Choosing from Various Optical Techniques ......................................... 9.1.13 Kцhler Illumination: Secrets of the Substage Condenser .......................................... 9.1.13 Oil Immersion ...................................... 9.1.16 Dark-Field, Rheinberg, Polarized-Light, Phase- Contrast, and DIC Microscopy ....................... 9.1.19 Acknowledgments .................................... 9.1.28 Literature Cited ................................... 9.1.29 Key References ..................................... 9.1.29 Internet Resources ................................. 9.1.29 9.2 Immunofluorescence Microscopy Overview and Principles ............................. 9.2.1 Strategic Planning .................................. 9.2.7 Safety Considerations .............................. 9.2.10 Protocols .......................................... 9.2.10 Basic Protocol 1: Processing Fibroblasts ........ 9.2.10 Basic Protocol 2: Processing Tetrahymena Cells .. 9.2.14 Alternate Protocol: Staining Cells Adhered to Poly-L-Lysine-Coated Coverslips .............. 9.2.15 Basic Protocol 3: Visualizing the Cells ......... 9.2.16 Reagents and Solutions ............................. 9.2.19 Understanding Results .............................. 9.2.20 Troubleshooting .................................... 9.2.21 Acknowledgments .................................... 9.2.22 Literature Cited ................................... 9.2.22 Internet Resources ................................. 9.2.23 10 Enzymatic Reactions 10.1 Working with Enzymes Introduction ....................................... 10.1.1 Overview of Enzymes ................................ 10.1.1 Handling Enzymes in the Laboratory ................. 10.1.6 Example of Setting Up an Enzymatic Reaction: Restriction Enzymes ............................... 10.1.16 Literature Cited .................................. 10.1.21 Key References .................................... 10.1.21 Internet Resources ................................ 10.1.21 10.2 Overview of PCR Overview and Principles ............................ 10.2.1 Strategic Planning ................................ 10.2.15 Strategic Questions ............................... 10.2.19 Safety Considerations ............................. 10.2.20 Protocols ......................................... 10.2.20 Basic Protocol: Routine PCR .................... 10.2.21 Support Protocol 1: Using Temperature Gradients for Rapid Optimization of PCR Cycling Conditions ............................. 10.2.26 Support Protocol 2: Titration of MgCh Concentration .................................. 10.2.26 Reagents and Solutions ............................ 10.2.27 Understanding Results ............................. 10.2.28 Troubleshooting ................................... 10.2.29 Variations ........................................ 10.2.29 Literature Cited .................................. 10.2.32 Internet Resources ................................ 10.2.35 10.3 Real-Time PCR Overview and Principles ............................ 10.3.1 Strategic Questions ................................ 10.3.5 Strategic Planning ................................. 10.3.7 Safety Considerations ............................. 10.3.26 Protocols ......................................... 10.3.26 Basic Protocol 1: Synthesis of cDNA by Reverse Transcription .......................... 10.3.27 Basic Protocol 2: Real-Time PCR Amplification and Analysis ................................... 10.3.28 Support Protocol 1: Determination of Amplification Efficiency ....................... 10.3.31 Support Protocol 2: Analyzing Results Using the Pfaffl Method to Calculate Fold Induction .. 10.3.32 Support Protocol 3: Serial Dilution for Standard Curve ................................. 10.3.34 Understanding Results ............................. 10.3.35 Troubleshooting ................................... 10.3.35 Variations ........................................ 10.3.38 Literature Cited .................................. 10.3.38 Internet Resources ................................ 10.3.40 11 Bioinformatics 11.1 Using NCBI BLAST Overview and Principles ............................ 11.1.1 Strategic Questions ................................ 11.1.5 Strategic Planning ................................. 11.1.5 Protocols .......................................... 11.1.9 Basic Protocol 1: Search a Nucleotide Database Using a Nucleotide Query: Nucleotide BLAST (BLASTN) ........................................... 11.1.9 Basic Protocol 2: Search a Protein Database Using a Protein Query: Protein BLAST (BLASTP) .. 11.1.12 Basic Protocol 3: Search a Protein Database Using a Translated Nucleotide Query: BLASTX .... 11.1.13 Basic Protocol 4: Searching a Translated Nucleotide Database Using a Protein or Nucleotide Query: TBLASTN and TBLASTX .......... 11.1.15 Understanding Results ............................. 11.1.16 Troubleshooting ................................... 11.1.24 Literature Cited .................................. 11.1.25 Internet Resources ................................ 11.1.26 Index